The baculoviruses are a family of large DNA viruses that are pathogenic to insects. The recent engineering of these viruses with novel toxin genes for improved properties as bio-pesticides has raised new concerns about host range and safety with respect to non-target organisms, including humans. Proper evaluation of baculovirus safety has demanded an increased understanding of the molecular mechanisms controlling host range and virus replication. The long term goal of this proposal is to examine the function and regulation of the earliest of baculovirus genes to define the mechanisms of host-virus interactions and virus replication strategies. We will focus primarily on the early 35K protein (p35) gene encoded by the prototype baculovirus, Autographa californica (multicapsid) nuclear polyhedrosis virus (AcMNPV). p35 is critical for virus replication in a cell-specific manner. It also prevents premature host cell lysis that is the result of virus-induced programmed cell death (apoptosis). Apoptosis is a built-in, signal-induced process by which a cell self-destructs; it is used to control cell numbers. Suppression of apoptosis results in aberrant cell survival and therefore contributes to oncogenesis. The mechanisms involved are unknown. AcMNPV is the first virus known to induce apoptosis in cells of non-immune origin and the first to simultaneously encode a gene (p35) for suppression of this host response. The ease with which p35 mutants can be generated and the relative convenience of culturing the responsive cells provide new opportunities to investigate the regulation of programmed cell death and to examine the possible role of apoptosis as an anti-virus defense strategy. In this proposal, we use a combination of site-directed mutagenesis and novel recombinant viruses to examine the mechanism by which p35 blocks apoptosis of AcMNPV-infected cells. Genetic and biochemical approaches will be used to explore the induction of apoptosis and its effect on replication, including virus DNA fragmentation and gene expression. We will also use the p35 gene as a sensitive reporter of early replication events in order to continue defining the cis-acting sequences and trans-acting factors (host and virus) involved in baculovirus transcriptional regulation. We focus here on the regulatory motifs in the upstream activating region of the promoter, the 5' noncoding RNA leader, and virus enhancers, each known to affect early transcription. Finally, having demonstrated that the p35 gene can be used as a powerful selectable marker (the first for baculoviruses), we develop a novel, transposon-based system for random mutagenesis of the AcMNPV genome that is critical for continued studies on virus gene organization and function. In summary, these studies are expected to yield important insight into the complex mechanisms of eukaryotic gene regulation as well as the induction and suppression of programmed cell death that is relevant to the control of cell proliferation.